Approximately one third of the E. coli and human proteomes consist of membrane proteins. Among these proteins are oxidoreductases, which fulfil central roles in the bioenergetic processes of cells, function as pathogenicity factors in microorganisms and their dysfunction is responsible for many inherited diseases in humans. They also find utility in industrial applications such as solar cells, biosensors and carbon sequestration. Unfortunately, many membrane-bound oxidoreductases, or the organisms in which they are found, are not easy to work with, and so our understanding of them relies on the ability to extrapolate from more easily tractable systems – Nitrate reductase (NarGHI) is such a system. NarGHI is a Molybdenum-containing enzyme expressed in E. coli when nitrate is available for anaerobic cellular respiration. Molybdoenzymes are widely found across the tree of life where they generally perform oxygen-transfer redox reactions. Bacterial respiratory diversity is largely due to the variety of molybdoenzymes they encode for, enabling a variety of terminal electron acceptors to be utilized for cellular respiration. Molybdoenzymes also largely facilitate the global biogeochemical Nitrogen cycle. Importantly, humans have four types of molybdoenzymes, which are active in drug metabolism, the catabolism of nucleotides, the processing of S-containing amino acids, and sulfite detoxification. Deficiencies in molybdoenzymes has serious health effects. The study of molybdoenzymes is therefore a critical and active area of research, for which NarGHI represents an excellent model system. Since it was first discovered, NarGHI has been keenly studied. It is not only an important enzyme in bacterial metabolism, but it is also a pathogenicity factor of certain microbes, and the principles of its structure and function are applicable to many human enzymes. NarGHI is a menaquinol:nitrate oxidoreductase that faces the cytoplasm from the plasma membrane. NarI is the membrane subunit in which quinol oxidation occurs, and NarG binds the molybdenum cofactor where nitrate is reduced. Quinol oxidation occurs at the periplasmic aspect of NarI and the electrons are transferred across the membrane via two b-type hemes. Electrons are subsequently transferred to NarG via NarH, which binds four iron-sulfur clusters. Overall, the electron transfer relay in NarGHI functions like a wire measuring ~100 Å long. This molecular wire feature is more the rule than the exception when it comes to integral membrane oxidoreductases. By understanding electron transfer in NarGHI, we can better understand transmembrane and long-distance electron transfer in numerous other enzymes. The process of quinol oxidation is also widely applicable to many enzymes in humans and bacteria, as quinone pool coupling is nearly universal and the basic principles of quinone chemistry are similar between disparate systems. However, while much has been learned about electron transfer and quinone binding in NarGHI, much remains to be understood. It has been the aim of this thesis to further probe the functioning of the membrane subunit, NarI. Specifically I sought to examine two key aspects of NarI function: 1) to probe the spectroscopic and electrochemical properties of the hemes and how these properties relate to transmembrane electron transfer; and 2) to gain a greater understanding of the mechanism by which NarI catalyses menaquinol oxidation. The findings of this thesis can be summarized in the following points: 1) The occupancy of the quinol oxidation site (Q-site) of NarI by quinones influences the EPR spectroscopic and electrochemical properties of the heme adjacent to the Q-site (heme bD). 2) The redox characteristics of the NarI hemes and the adjacent iron-sulfur cluster of NarH facilitate transmembrane electron transfer in a controlled manner. 3) Quinol oxidation proceeds via a protonated intermediate, where its binding to NarI requires the Q-site be deprotonated. 4) The deprotonation of inbound quinol is catalysed by a partially conserved lysine (Lys86), and that the property of Lys86 that is most important is its ionisability. 5) The Q-site of NarI requires that Lys86 undergo a conformational change in order to facilitate quinol deprotonation. This is hinted at by an engineered second-site rescuer mutation which restores quinol oxidase activity in a catalytically dead variant of Lys86 (Lys86Ala).

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